Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Synthesis of peptides
Reexamination Certificate
1998-08-05
2001-06-05
Jones, Dwayne C. (Department: 1654)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Synthesis of peptides
C530S328000, C530S335000, C530S336000, C530S338000, C530S339000
Reexamination Certificate
active
06242565
ABSTRACT:
The present invention relates to a method for preparing a peptide derivative having physiological activities, and more particularly to a method for preparing a peptide derivative which is useful for the prevention of early abortion and which exhibits anti-oxytocin activities while exhibiting reduced anti-vasopressin activities. The invention also relates to intermediates useful in the method, as well as to the method for preparing the intermediates.
Pre-full-term delivery is one of the most serious problems in obstetrics. Many pregnant women who have passed 20 weeks of gestation have experienced premature births and immature deliveries, which together constitute the primary cause of neonatal diseases and neonatal deaths. Therefore, even today, when neonatal treatment is greatly advanced, the consensus opinion is that the fetus is preferably carried by its mother's body until the month of expected delivery.
Uterine muscle relaxants are generally used for the treatment of pre-full-term delivery. In recent years, oxytocin antagonists have been proposed as ideal uterine muscle relaxants for suppressing pre-full-term delivery. This proposal was based on the finding that oxytocin, a peptide hormone, is a physiological parturifacient. Further research led to the finding of a novel oxytocin receptor antagonist which is a peptide derivative of the following formula (I):
and which is disclosed in International Patent Publication WO94/25485.
Oxytocin, a posterior pituitary hormone, is composed of nine amino acid residues. Vasopressin, another posterior pituitary hormone, is also composed of nine amino acid residues, which with the exception of two amino acid residues are identical to those of oxytocin. These two substances, notwithstanding their similar structures, exhibit very different physiological activities; oxytocin functions to constrict smooth muscles including uterine muscle, whereas vasopressin exhibits peripheral vasoconstriction and elevation of blood pressure. Thus, anti-oxytocin drugs previously developed often exhibit a side effect of anti-vasopressin activity.
However, the aforementioned peptide derivative of formula (I) are considered to be ideal uterine muscle relaxants, because the derivatives function as potent oxytocin receptor antagonists while they never exhibit vasopressin antagonizing activity, which is a side effect exerted by conventional oxytocin receptor antagonists.
WO94/25485 referred to hereinabove discloses a method for preparing the compounds of formula (I). According to that publication, the peptide derivatives are prepared by use of a so-called solid phase method in which a resin carrier is employed. Mass-production of the compounds of formula (I) at high purity and high yield is difficult to achieve by the solid phase method.
For example, as experienced with commercially available automatic peptide synthesizers, which automatically synthesize peptides making use of the solid phase method, the process steps are lengthy, because amino acids are sequentially bonded to the resin carrier in a one-by-one manner through condensation reaction. It is also noted that in each step, post-condensation purification cannot be performed, and inclusion of peptides that lack portions of amino acids cannot be prevented. In order to reduce the occurrence of such defective peptides, condensation reaction is performed by use of an excessive amount of an amino acid derivative, i.e., 2 to 6 times that of the amino group content of peptide joined to the carrier resin. However, under general circumstances, prevention of the occurrence of defective peptides by such an approach is difficult.
It is also noted that in a step in which a required peptide moiety is separated from the carrier resin, contamination is caused by the side reaction products on the cleavage of peptide resins and also by impurities such as defective peptides that lack one or more amino acids, the defective peptides being formed because the intermediate cannot be purified in the course of reaction. Therefore, an intricate purification process must be performed to obtain the target compound having a high level of purity.
Moreover, according to the above-mentioned publication, the target peptide is separated from the carrier resin by use of a liquid ammonia solution in which sodium metal is dissolved, and such a method is industrially difficult to perform.
In the field of peptide synthesis, many methods have been disclosed for forming intramolecular SS bonds (“Development of Pharmaceuticals, Second Series” Vol. 14, ‘Peptide Synthesis’ pp. 233-258, 1991, Hirokawa Shoten). Among such methods, when potassium ferricyanide is used, the reaction is usually performed in the vicinity of neutral pH, i.e., between 6.0 and 8.0 (Chem. Pharm. Bull., Vol. 38, page 1920, 1990). However, under this reaction condition, intermolecular SS bonds are formed in much greater numbers than are intramolecular SS bonds or intramolecular cyclization, and therefore, the target compound cannot be obtained at high yield.
In addition, according to the method disclosed in the aforementioned publication WO94/25485, the intermediate used in the synthesis of the formula (I) compound is a compound in which the protective group for thiol is 4-methylbenzyl. Accordingly, when an intramolecular SS bond (disulfide bond) is formed, great care must be taken so as to assure safety, etc., because liquid ammonia solution in which metallic sodium has been dissolved is employed for removing the protective group.
Accordingly, the general object of the present invention is to provide a compound of formula (I) at high yield and a high level of purity in an industrially efficient manner.
The present inventors have conducted extensive studies on methods for preparing peptide derivatives of formula (I), and have successfully established a synthesis scheme which is not dependent on the solid phase method and is suited to the liquid phase method, and have found that the above-mentioned general object can be achieved by use of a certain intermediate, which is cyclized via the formation of intramolecular SS bonds through a specific process, thus leading to completion of the present invention.
The present invention provides a method for preparing a peptide derivative of formula (I) or a salt thereof:
which method comprises the steps of removing two MBzl groups from a compound of formula (II) or a salt thereof
wherein MBzl represents a 4-methoxybenzyl group which serves as a protective group for a thiol group, R
1
represents a hydrogen atom or a protective group for the indolyl group of D-Trp and R
2
represents a hydrogen atom or a protective group for the guanidino group of Arg, and subsequently oxidizing in an aqueous medium having a pH from 4 to 6 to form an intramolecular disulphide (SS) bond.
The present invention also provides a compound of formula (II) or a salt thereof as described above, which serves as an intermediate in the synthesis of the formula (I) compound.
The present invention further provides a method for preparing a compound of formula (III) or a salt thereof: wherein R
1
represents a hydrogen atom or a protective group for the indolyl group of D-Trp, R
2
represents a
hydrogen atom or a protective group for the guanidino group of Arg, and R
3
is a protective group for a thiol group; which method comprises reacting a compound of formula (Xa) or a salt thereof:
wherein R
1
, R
2
and R
3
are as defined above; with a compound of formula (XI):
wherein R
3
is a protective group for thiol to obtain a compound of formula (III) or a salt thereof.
In addition, the present invention provides a compound of formula (III) or a salt thereof as described above, which serves as an intermediate in the synthesis of formula (I) compound.
REFERENCES:
patent: 4719199 (1988-01-01), Yim
patent: 5373089 (1994-12-01), Flouret et al.
patent: 5596078 (1997-01-01), Andersson et al.
patent: 5712418 (1998-01-01), Carpino et al.
patent: WO 94/25485 (1994-11-01), None
Manning, M. et al: “Effects of a D-Cys6/L-Cys6 Interchange in Nonselective and Selective Vasopre
Kimura Hitoshi
Kishida Satoshi
Nakanishi Akihiro
Delacroix-Muirheto C.
Jones Dwayne C.
Nixon & Vanderhye P.C.
TT Pharmaceuticals, Inc.
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